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='''Testing human 18S using regular PCR'''= ==Objective== #Check if the 18S primers are specific by agarose gel. #Using different conc. of human gDNA to optimize the condition. #Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened. ==Reagents== #H2O - (Ambion) #Enzyme - Taq 2X master mix (New England Biolabs) #Template - diluted human genome DNA (Jurkat cell, New England Biolabs #18S primer mix (10 uM Forward + 10 uM Reverse) -> hS18_212, hS18_306 (10 uL 100 uM Forward + 10 uL 100 uM Reverse + 80 uL H2O) ==Exp. Design== Template gDNA (100 ng/uL) H2O ------------------------------ 1/10 1/100 1/1000 Dilution factor ------------------------------ Primer: 18S-211 bp #1 #2 #3 #4 (#1, #2 - replicates) 18S-306 bp #5 #6 #7 #8 (#4, #5 - replicates) ==Preparation== '''gDNA Template dilution''': Stock solution 100 ug/mL = 100 ng/uL Oligo tube Dilution from 100 ng/uL ->10 ng/uL (1/10), 1 ng/uL (1/100), 100 pg/uL (1/1000) ==Procedures== Step1: Preparing master mix 1 8+2 rxn -------------------------------------------- H2O 8.0 80.0 uL Taq 2X Master Mix 10.0 100.0 uL -------------------------------------------- 18.0 180.0 uL(180/10=18) Step2: Transfer 18 uL of Master mix into each of 8 wells in PCR tube-strip Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design Step4: Perform regular PCR Machine: Bio-Rad 2 blocks- DNA engine Program name: Gene59 94C 2min -> (94C 40 sec -> 59C 40 sec -> 72C 1 min ) x 29 cycle -> 72C 10 min -> 4C Forever Step5: Agarose gel electrophoresis condition 1.5% agarose gel: 0.75 g + 50 mL (0.5 TBE). Heat up with 30 sec + 20 sec Gel stain: 4uL SYBR safe (Invitrogen)/ 50 mL Agarose gel mix Small gel tray with 8-well comb Ladder: Low mass ladder (Invitrogen) Sample loading: (6 uL sample + 1 uL dye) -> load 6 uL mix Loading order: L->R, #1~#8 (02-05-09' PCR products) -> #1~#6(02-04-09' PCR products) -> ladder Running at 135 V for 30 min. ==Results== '''FIG. Gel picture of 18S primers testing''' [[Image:ZhangLab_Gel pic 020609-new.jpg|600px|none]] #02-04-09' results: PCR products #1 and #2 - duplicates (primer S18_211, amplicon 211 bp) , #4 and #5 - duplicates (primer S18_306, amplicon 306 bp), #3 and #6 blank (no template control) #02-05-09' results: PCR products #1, #2 and #3 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_211, amplicon 211 bp) , #4 - blank; #5, #6 and #7 - template (10 ng, 1 ng, 100 pg respectively. Primer S18_306, amplicon 306 bp) , #8 - blank ==Discussion== #The band specificy is acceptible for both S18 primers, and no contaminatino in reagents was found. #Both condition of both realtime PCR and coventional PCR were working and appliable for following experiments. Both EconoTaq 2X Master and NEB Taq 2X Master work well in these amiplifications. #The results from 02-04-09' indicated that realtime PCR reaction was acturally successful for 20-cycle amplification. It's unknow why the real-time monitoring of signal curve was shown correctly on the Chromo4 machine. (The SYBR 2X didn't fucntion?) #The results from 02-05-09' indicated that the dilution of template is effective to be seen on agarose gel. ==Suggestion== #prepared a new tube of SYBR-2X and test it with old SYBR-2X regent using a small-scale realtime PCR reaction.
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